This invention is in the field of plant virology. Specifically, the invention relates to the synthesis of nucleic acid sequences encoding an altered viral movement protein, construction of viral vectors expressing such protein, and generation of host plants infected by the viral vectors. The viral vectors permit rapid local and systemic invasion of a host, and allow stable expression of a transgene of interest.
In the last fifteen years, considerable progress has been made in expressing foreign genes in plants. Foreign proteins are now routinely produced in many plant species for modification of the plant or for production of proteins for use after extraction. Vectors for the genetic manipulation of plants have been derived from several naturally occurring plant viruses. For the production of specific proteins, transient expression of foreign genes in plants using virus-based vectors has several advantages. Products of plant viruses are among the highest produced proteins in plants. Often a viral gene product is the major protein produced in plant cells during virus replication. Many viruses are able to systemically move from an initial infection site to almost all cells of the plant. Because of these reasons, plant viruses have been developed into efficient transient expression vectors for foreign genes in plants. Viruses of multi-cellular plants are relatively small, probably due to the size limitation in the pathways that allow viruses to move to adjacent cells in the systemic infection of entire plants. One such plant virus upon which plant expression vectors are based is TMV (tobacco mosaic virus). TMV is the type member of the tobamovirus group. TMV has straight tubular virions of approximately 300xc3x9718 nm with a 4 nm-diameter hollow canal consisting of approximately 2000 units of a single capsid protein wound helically around a single RNA molecule. Virion particles are 95% protein and 5% RNA by weight. The genome of TMV is composed of a single-stranded RNA of 6395 nucleotides containing five large ORFs. Expression of each gene is regulated independently. The virion RNA serves as the messenger RNA (mRNA) for the 5xe2x80x2 genes, encoding the 126 kDa replicase subunit and the overlapping 183 kDa replicase subunit that is produced by read through of an amber stop codon approximately 5% of the time. Expression of the internal genes is controlled by different promoters on the minus-sense RNA that direct synthesis of 3xe2x80x2 -coterminal subgenomic mRNAs which are produced during replication. A detailed description of tobamovirus gene expression and life cycle can be found, among other places, in Dawson and Lehto, Advances in Virus Research38:307-342(1991).
Thus, it is of scientific and commercial interest to provide new and improved vectors for the genetic manipulation of plants.
A principal aspect of the present invention is the design of a recombinant viral vector expressing an altered movement protein and altered 126/183 viral proteins to affect stable expression of a transgene in a plant host.
Accordingly, the present invention provides an isolated nucleic acid sequence encoding an altered viral movement protein having the amino acid sequence shown in SEQ ID NOS.: 5 and 6 and altered 126/183 viral proteins. In one aspect, the isolated nucleic acid sequence is essentially identical to the sequence shown in SEQ ID NOS.: 3 and 4, and it contains a Thymine (T) or Uracil (U) residue at position 5213 and Guanine (G) residue at 5303 as shown in FIG. 1A. In another aspect, the isolated nucleic acid sequence is identical to the sequence shown in SEQ ID NOS.: 3 and 4. The alteration of the 30K movement protein and alteration of the 126/183 viral proteins results in an enhanced ability to facilitate stabilization of a transgene contained in a viral vector.
In a separate embodiment, the present invention provides a viral vector comprising the nucleic acid sequence encoding an altered viral movement protein having the amino acid sequence shown in SEQ ID NOS.: 5 and 6 and altered 126/183 viral proteins. In one aspect, the viral vector exhibits an enhanced ability compared to a control viral vector to stabilize a transgene contained in the vector. Preferably, the vector is a tobacco mosaic viral vector. A particularly preferred vector is designated BSG1057 (deposited with American Type Culture Collection having accession number 20398, which was deposited on Apr. 28, 1999).
In a separate aspect within this embodiment, the viral vector comprises a transgene of interest. Preferably the transgene is a non-viral gene encoding a protein selected from the group consisting of a membrane protein, a cytosolic protein, a secreted protein, a nuclear protein, and a chaperon protein.
The present invention also provides a cell transformed with a subject viral vector. The transformed cell may be animal or plant. Preferably, the cell is a plant cell. The present invention further provides a transgenic plant comprising the viral vector. Preferred transgenic plant may, for example, be Nicotiana benthamiana or Nicoliana tabacum, but others may be just as readily substituted by one of skill in the art.